System and method for secure document printing and detection

ABSTRACT

A method for authenticating and verifying an item to be genuine is described. The method for authenticating the item comprises applying a particular nucleic acid material associated with a particular sequence of nucleic acid bases to ink within an ink cartridge or a toner compound within a toner housing. The method also comprises collecting a sample of either the ink or toner compound and verifying the ink or toner is genuine by detecting the particular nucleic acid material.

CROSS REFERENCE

This application is a continuation-in-part of patent application Ser.No. 11/437,265 having a filing date of May 19, 2006 that is related toprovisional patent application 60/682,976 filed on May 20, 2005; thisapplication is also a continuation-in-part of patent application Ser.No. 10/825,968 having a filing date of Apr. 15, 2004 that is related toprovisional patent application 60/463,215 filed on Apr. 16, 2003; thisapplication is also related to provisional patent application 60/874,425having a filing date of Dec. 12, 2006; this application is also relatedto provisional patent application 60/877,875 having a filing date ofDec. 29, 2006; this application is also related to provisional patentapplication 60/877,869 having a filing date of Dec. 29, 2006; each ofthe patent applications being hereby incorporated by reference includingco-pending patent application Ser. No. ______ filed on Dec. 11, 2007.

FIELD

This invention relates to a system and method for secure documentprinting and detection. More particularly, the invention is related to asystem and method for secure document printing and detection ofcounterfeit ink cartridges and laser toner.

BACKGROUND

With the dawn of the information age comes the ability to duplicate,change, alter and distribute just about anything. Law enforcementorganizations have called counterfeiting the crime of the 21^(st)century. Product counterfeiting is a serious and growing threat.Measures to defend against counterfeiters are being taken by manycorporations, but they have not developed comprehensive, systematic, andcost-effective solutions to preventing counterfeiting.

Due to advancing counterfeiting techniques, traditional anti-counterfeittechnologies are becoming obsolete. Additionally, governments andcorporations that have invested a great deal of resources in fightingcounterfeiting have experienced little success. Furthermore, lawenforcement agencies that are burdened with efforts to combat violentcrimes have insufficient resources to fight the “victimless”counterfeiting crime.

Counterfeiting also extends to specific products where consumables areindispensable for the continued use of a product. For example, itemssuch as an ink cassettes or cartridges must be replaced several timesduring the life of a laser or ink jet printer. Counterfeiting of thisnature is particular adverse to the interests of original manufacturersof the product because not only does this negatively impact the sales oftheir original consumables, the use of a counterfeit cartridge, forexample, may severely damage the printer over time, effectivelyimpairing it from working properly. Consequently, both the public andthe manufacturer face non-trivial consequences due to the widespreadavailability of counterfeit consumable items because any user may besold counterfeit consumable items unbeknownst to the consumer.

SUMMARY

This invention relates to methods for authenticating inks, paints,pigments, ink within ink cartridges, or items with such ink printed ortransferred thereon. The invention utilizes compositions which linkbiomolecules to visual or machine-detectable reporters. The methods ofauthentication comprise placing, associating, or integrating an opticalreporter taggant with the ink, ink cartridge, printed item or other itemof interest. The optical reporters can be easily detected by using ahigh energy light source for excitation, with the location of labeledbiomolecules identified by the presence of an optical reporter. Thelocation and emission wavelength of the optical reporters provides afirst level of security or authentication of the tagged item ofinterest. After the location of the optical reporters and associatedbiomolecules on the item has been determined, the biomolecules may becharacterized and identified to further increase the level of securityand/or authenticity of the item. When the biomolecule attached to theoptical reporter is a DNA molecule, PCR or sequence analysis techniquescan be utilized to further authenticate the item.

In one embodiment the invention provides an authentication method forauthenticating an ink within an ink cartridge, the method comprising:applying a particular nucleic acid material associated with a particularsequence of nucleic acid bases to the ink; collecting a sample of theink having the nucleic acid; and verifying whether the ink is genuine bydetecting said particular nucleic acid material.

The particular nucleic acid material may, in certain embodiments, bedeoxy-ribo nucleic acid (DNA). In other embodiments the particularnucleic acid material may be ribonucleic acid (RNA).

In certain embodiments the method further comprises detecting theparticular nucleic acid by performing a polymerase chain reaction (PCR)of the nucleic acid material.

In certain embodiments, the method for authenticating ink in an inkcartridge comprises the steps of, providing an optical reporter marker,the optical reporter marker having at least one light emittingupconverting phosphor particle linked to at least one nucleic acidmaterial, the nucleic acid material having an identifiable portion,introducing the optical reporter marker to the ink of interest, thendetecting the optical reporter marker associated with the ink with alight source, obtaining a sample of the optical reporter marker from theink of interest for analysis; followed by analyzing the collected sampleto detect the presence of the identifiable portion of the nucleic acidmaterial linked to the upconverting phosphor particle.

This invention relates to methods for authenticating an that utilizecompositions which link biomolecules to visual or machine-detectablereporters. The methods of authentication comprise placing, associating,or integrating an optical reporter taggant with the ink. The opticalreporters can be easily detected by using a high energy light source forexcitation, with the location of labeled biomolecules identified by thepresence of an optical reporter. The location and emission wavelength ofthe optical reporters provides a first level of security orauthentication of the tagged ink. After the location of the opticalreporters and associated biomolecules on the document has beendetermined, the biomolecules may be characterized and identified tofurther increase the level of security and/or authenticity of the ink.When the biomolecule attached to the optical reporter is a DNA molecule,PCR or sequence analysis techniques can be utilized to furtherauthenticate the ink.

In many embodiments of the method for authenticating an ink comprisesthe steps of;

-   -   providing an optical reporter marker, the optical reporter        marker having at least one light emitting upconverting phosphor        particle linked to at least one nucleic acid taggant, the        nucleic acid taggant having an identifiable portion,    -   introducing the optical reporter marker to the ink,    -   detecting the optical reporter marker associated with the ink        with a light source,    -   obtaining or collecting a sample of the optical reporter marker        from the ink for analysis; and    -   analyzing the collected sample to detect the presence of the        identifiable portion of the nucleic acid taggant linked to the        upconverting phosphor particle. In many embodiments the        analyzing of the collected sample comprises determining the DNA        sequence of the nucleic acid taggant, and comparing the        determined DNA sequence with a known or reference DNA sequence.

In many embodiments, the optical reporter marker has the composition ofthe formula I:

(cOpR)-[L-(NA)]_(m)  I

wherein:

-   -   m is an integer greater than 1;    -   (cOpR) is a coated optical reporter particle;    -   (NA) is a nucleic acid oligomer of the nucleic acid material of        detectable sequence; and    -   L is a linking group covalently bound to the coated optical        reporter particle and to the nucleic acid oligomer.

In some embodiments, (cOpR) comprises an upconverting phosphor (UCP)material.

The (cOpR) of the composition may comprise an upconverting phosphor(UCP) material coated with silica. Where the compositions are coatedwith silica, the silica comprises at least one Si—O bond.

In most embodiments (NA) is a single or double stranded DNA moleculehaving a length of between about 40 base pairs and about 1000 basepairs.

The linker L of the composition may comprise an alkylene moiety having afirst end covalently bound to the coated optical reporter particle and asecond end covalently bound to the nucleic acid oligomer.

Where the composition utilized in the methods of the invention comprisesa (UCP), in certain embodiments, the (UCP) is an upconverting phosphorparticle of the formula:

Y_(x)Yb_(y)Er_(z)O₂S; or

Na(Y_(x)Yb_(y)Er_(z))F₄;

wherein:

-   -   x is from about 0.6 to about 0.95;    -   y is from about 0.05 to about 0.35; and    -   z is from about 0.1 to about 0.001.

In other embodiments, the linker L may be of the formula:

-A-R¹—B—

where R¹ is C₂₋₈alkylene, -A- is a group covalently bonded to thesurface of the coated optical reporter and —B— is a group covalentlybonded to the 3′ or 5′ end of the nucleic acid oligomer.

In other embodiments, a composition used in the methods forauthenticating an ink of the invention has the formula:

(UCP)-[A-R¹—B-(DNA)]_(m)

where m is an integer greater than 1; UCP is an upconverting phosphorparticle; DNA is a single or double stranded deoxyribonucleic acidoligomer; R¹ is C₂₋₈alkylene; -A- is a group capable of covalentlybonding to the surface of the upconverting phosphor particle and —B— isa group capable of bonding to the 3′ or 5′ end of the deoxyribonucleicacid oligomer.

All patents and publications identified herein are incorporated hereinby reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of one embodiment of the methods of theinvention.

FIG. 2 is a flow chart of one embodiment of the methods forauthenticating an item in accordance with the invention.

FIG. 3 is a perspective-view of a printer.

FIG. 4 is a perspective-view of a printer carriage and its ink tonercartridge.

FIG. 5 is a plot of a real-time PCR results for a composition of theinvention, comprising an optical reporter linked to a sequenceable DNAmolecule.

DESCRIPTION

Before the present methods for authenticating products are described, itis to be understood that this invention is not limited to particularproduct described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “ataggant” includes a plurality of such taggants and reference to “theprimer” includes reference to one or more primers and equivalentsthereof known to those skilled in the art, and so forth.

If any publications are discussed here, they are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention. Further, the dates of publication provided may bedifferent from the actual publication dates which may need to beindependently confirmed.

Although the description about the methods for verifying authenticity ofan item contains many limitations in the specification, these should notbe construed as limiting the scope of the claims but as merely providingillustrations of some of the presently preferred embodiments of thisinvention. Many other embodiments will be apparent to those of skill inthe art upon reviewing the description. Thus, the scope of the inventionshould be determined by the appended claims, along with the full scopeof equivalents to which such claims are entitled.

DEFINITIONS

Unless otherwise stated, the following terms used in this Application,including the specification and claims, have the definitions givenbelow. It must be noted that, as used in the specification and theappended claims, the singular forms “a”, “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

“Alkyl” means the monovalent linear or branched saturated hydrocarbonmoiety, consisting solely of carbon and hydrogen atoms, having from oneto twelve carbon atoms. “Lower alkyl” refers to an alkyl group of one tosix carbon atoms, i.e. C₁-C₆alkyl. Examples of alkyl groups include, butare not limited to, methyl, ethyl, propyl, isopropyl, isobutyl,sec-butyl, tert-butyl, pentyl, n-hexyl, octyl, dodecyl, and the like.

“Alkenyl” means a linear monovalent hydrocarbon radical of two to sixcarbon atoms or a branched monovalent hydrocarbon radical of three tosix carbon atoms, containing at least one double bond, e.g., ethenyl,propenyl, and the like.

“Alkynyl” means a linear monovalent hydrocarbon radical of two to sixcarbon atoms or a branched monovalent hydrocarbon radical of three tosix carbon atoms, containing at least one triple bond, e.g., ethynyl,propynyl, and the like.

“Alkylene” means a linear saturated divalent hydrocarbon radical of oneto six carbon atoms or a branched saturated divalent hydrocarbon radicalof three to six carbon atoms, e.g., methylene, ethylene,2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene,and the like.

“Alkoxy” and “alkyloxy”, which may be used interchangeably, mean amoiety of the formula —OR, wherein R is an alkyl moiety as definedherein. Examples of alkoxy moieties include, but are not limited to,methoxy, ethoxy, isopropoxy, and the like.

“Alkoxyalkyl” means a moiety of the formula R^(a)—O—R^(b)—, where R^(a)is alkyl and R^(b) is alkylene as defined herein. Exemplary alkoxyalkylgroups include, by way of example, 2-methoxyethyl, 3-methoxypropyl,1-methyl-2-methoxyethyl, 1-(2-methoxyethyl)-3-methoxypropyl, and1-(2-methoxyethyl)-3-methoxypropyl.

“Alkylcarbonyl” means a moiety of the formula —R′—R″, where R′ is oxoand R″ is alkyl as defined herein.

“Alkylsulfonyl” means a moiety of the formula —R′—R″, where R′ is —SO₂—and R″ is alkyl as defined herein.

“Alkylsulfonylalkyl means a moiety of the formula —R′—R″—R″″ where R′ isalkylene, R″ is —SO₂— and R″″ is alkyl as defined herein.

“Amino means a moiety of the formula —NR—R′ wherein R and R′ eachindependently is hydrogen or alkyl.

“Alkylsulfanyl” means a moiety of the formula —SR wherein R is alkyl asdefined herein.

“Urea” or means a group of the formula —NR′—C(O)—NR″R″″ wherein R′, R″and R″″ each independently is hydrogen or alkyl.

“Carbamate” means a group of the formula —O—C(O)—NR′R″ wherein R′ and R″each independently is hydrogen or alkyl.

“Carboxy” means a group of the formula —O—C(O)—OH.

“Sulfonamido” means a group of the formula —SO₂—NR′R″ wherein R′, R″ andR″″ each independently is hydrogen or alkyl.

“Optionally substituted”, when used in association with “aryl”, phenyl”,“heteroaryl” “cycloalkyl” or “heterocyclyl”, means an aryl, phenyl,heteroaryl, cycloalkyl or heterocyclyl which is optionally substitutedindependently with one to four substituents, preferably one or twosubstituents selected from alkyl, cycloalkyl, cycloalkylalkyl,heteroalkyl, hydroxyalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino,acylamino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy,heteroalkyl, —COR, —SO₂R (where R is hydrogen, alkyl, phenyl orphenylalkyl), —(CR′R″)_(n)—COOR (where n is an integer from 0 to 5, R′and R″ are independently hydrogen or alkyl, and R is hydrogen, alkyl,cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), or—(CR′R″)_(n)—CONR^(a)R^(b) (where n is an integer from 0 to 5, R′ and R″are independently hydrogen or alkyl, and R^(a) and R^(b) are,independently of each other, hydrogen, alkyl, cycloalkyl,cycloalkylalkyl, phenyl or phenylalkyl).

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not.

“Inert organic solvent” or “inert solvent” means the solvent is inertunder the conditions of the reaction being described in conjunctiontherewith, including for example, benzene, toluene, acetonitrile,tetrahydrofuran, N,N-dimethylformamide, chloroform, methylene chlorideor dichloromethane, dichloroethane, diethyl ether, ethyl acetate,acetone, methyl ethyl ketone, methanol, ethanol, propanol, isopropanol,tert-butanol, dioxane, pyridine, and the like. Unless specified to thecontrary, the solvents used in the reactions of the present inventionare inert solvents.

“Solvates” means solvent additions forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

The term “emitting reporter” means a chemical substituent or materialthat produces, under appropriate excitation conditions, a detectableoptical signal. The optical signal produced by an emitting reporter istypically electromagnetic radiation in the near-infrared, visible, orultraviolet portions of the spectrum. The emitting reporters of theinvention are generally up-converting reporters, but can also be forexample, fluorescent and calorimetric substituents.

The term “phosphor particle” means a particle or composition comprisingat least one type of upconverting phosphor material.

The term “primer” means a nucleotide with a specific nucleotide sequencewhich is sufficiently complimentary to a particular sequence of a targetDNA molecule, such that the primer specifically hybridizes to the targetDNA molecule.

The term “probe” refers to a binding component which bindspreferentially to one or more targets (e.g., antigenic epitopes,polynucleotide sequences, macromolecular receptors) with an affinitysufficient to permit discrimination of labeled probe bound to targetfrom nonspecifically bound labeled probe (i.e., background).

The term “probe polynucleotide” means a polynucleotide that specificallyhybridizes to a predetermined target polynucleotide.

The term “oligomer” refers to a chemical entity that contains aplurality of monomers. As used herein, the terms “oligomer” and“polymer” are used interchangeably. Examples of oligomers and polymersinclude polydeoxyribonucleotides (DNA), polyribonucleotides (RNA), otherpolynucleotides which are C-glycosides of a purine or pyrimidine base,polypeptides (proteins), polysaccharides (starches, or polysugars), andother chemical entities that contain repeating units of like chemicalstructure.

The term “PCR” refers to polymerase chain reaction. This refers to anytechnology where a nucleotide is amplified via a temperature cyclingtechniques in the presence of a nucleotide polymerase, preferably a DNApolymerase. This includes but is not limited to real-time PCRtechnology, reverse transcriptase-PCR, and standard PCR methods.

The term “nucleic acid” means a polymer composed of nucleotides, e.g.deoxyribonucleotides or ribonucleotides, or compounds producedsynthetically which can hybridize with naturally occurring nucleic acidsin a sequence specific manner analogous to that of two naturallyoccurring nucleic acids, e.g., can participate in hybridizationreactions, i.e., cooperative interactions through Pi electrons stackingand hydrogen bonds, such as Watson-Crick base pairing interactions,Wobble interactions, etc.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymercomposed of ribonucleotides.

The terms “deoxy-ribonucleic acid” and “DNA” as used herein mean apolymer composed of deoxyribonucleotides.

The term “polynucleotide” or “nucleotide” refer to single or doublestranded polymer composed of nucleotide monomers of generally greaterthan 50 nucleotides in length.

The term “monomer” as used herein refers to a chemical entity that canbe covalently linked to one or more other such entities to form anoligomer. Examples of “monomers” include nucleotides, amino acids,saccharides, peptides, and the like. The term nucleotide means

The term “linker” means a compound or a composition which covalentlylinks a biomolecule to the surface of a coated emitting reporter. Forexample, but not limited to a silyinated coated upconverting phosphorparticle linked to a DNA molecule.

The term “identifiable sequence” or “detectable sequence” means anucleotide sequence which can by detected by hybridization and/or PCRtechnology by a primer or probe designed for specific interaction withthe target nucleotide sequence to be identified. The interaction of thetarget nucleotide sequence with the specific probe or primer can bedetected by optical and/or visual means to determine the presence of thetarget nucleotide sequence.

A “Nucleic acid tag” is a nucleic acid oligomer or fragment used toidentify or authenticate a particular product. Nucleic acid tag andnucleic acid taggant are interchangeable throughout the specification.

The term “DNA taggant” means a nucleic acid tag which comprises deoxynucleotides. A DNA taggant maybe double stranded or single stranded,cDNA, STR (short tandem repeats) and the like. The DNA taggant may alsocomprise modification to one or more nucleotides which aid in theidentification or detection of the DNA taggant.

The term “DNA marker compound” means a marker compound utilized toidentify or authenticate a particular product which comprises a specificDNA oligomer which is used to authenticate the particular product.

The terms “those defined above” and “those defined herein” whenreferring to a variable incorporates by reference the broad definitionof the variable as well as preferred, more preferred and most preferreddefinitions, if any.

Nomenclature and Structures

In general, the nomenclature used in this Application is based onAUTONOM™ v.4.0, a Beilstein Institute computerized system for thegeneration of IUPAC systematic nomenclature. Chemical structures shownherein were prepared using ISIS® version 2.5. Any open valency appearingon a carbon, oxygen sulfur or nitrogen atom in the structures hereinindicates the presence of a hydrogen atom unless indicated otherwise.Where a chiral center exists in a structure but no specificstereochemistry is shown for the chiral center, both enantiomersassociated with the chiral center are encompassed by the structure.Where a structure shown herein may exist in multiple tautomeric forms,all such tautomers are encompassed by the structure.

A method for securing an item and verifying its authenticity as genuineby combining an object or product with a specified nucleic acid tag andthen detecting the nucleic acid tag in the object or product in aneffective manner is described. FIG. 1 shows a flow chart of the generalprocess 100 of introducing a nucleic acid tag into or onto an item andbeing able to detect the nucleic acid tag or marker incorporated in theitem. The process comprises applying at least one specific nucleic acidfragment, as an authentication tag or marker for a product in step 102.The nucleic acid (NA) marker may be DNA, cDNA, or other DNA material, orany other nucleic acid fragment comprising nucleic acids or nucleic acidderivatives. The marker may be a nucleic acid fragment that is singlestranded or preferably, double stranded and may vary in length,depending on the product to be labeled as well as the detectiontechnique utilized in the nucleic acid marker detection process.

The nucleic acid marker may be synthetically produced using a nucleicacid synthesizer or by isolating nucleic acid material from yeast, humancell lines, bacteria, animals, plants and the like. In certainembodiments, the nucleic acid material may be treated with restrictionenzymes and then purified to produce an acceptable nucleic acidmarker(s). The length of the nucleic acid marker/tag usually rangesbetween about 100 to about 10 kilo bases, more usually about 500 basesto about 6 kb, and preferably about 1 kb to about 3 kb in length.

The nucleic acid taggant may comprise one specific nucleic acid sequenceor alternatively, may comprise a plurality of various nucleic acidsequences. In one embodiment, polymorphic DNA fragments of the typeshort tandem repeats (STR) or single nucleotide polymorphisms (SNP) areutilized as an anti-counterfeit nucleic acid tag. While the use of asingle sequence for a nucleic acid marker may make detection of themarker easier and quicker, the use of a plurality of nucleic acidsequences such as STR and SNP, in general, give a higher degree ofsecurity against forgers.

For exemplary purposes, the nucleic acid concentration may vary frompico grams (1×10⁻¹² gram) to micro grams (1×10⁻⁹ gram).

In certain embodiments of the methods of the invention, the nucleic acidmarker is derived from DNA extracted from a specific plant source and isspecifically digested and ligated to generate artificial nucleic acidsequences which are unique to the world. The digestion and ligation ofthe extracted DNA is completed by standard restriction digestion andligase techniques known to those skilled in the art of molecularbiology. Once the modified DNA taggant has been produced, the taggant isencapsulated into materials for protection against UV and degradation.The DNA encapsulant materials are generally of plant origin.

The marker compound maybe produced as a solid or liquid, water or oilbased, a suspension, an aggregate and the like. One feature of themarker compounds is to protect the nucleic acid fragment from UV andother degradation factors that may degrade the nucleic acid taggantovertime, while the nucleic acid is acting as an authentication tag fora particular product. In certain embodiments, when the taggant is DNA,the nucleic acid tag may be encapsulated and suspended in a solventsolution (aqueous or organic solvent solution) producing a “stock” DNAtaggant solution at a specified concentration. This stock DNA solutioncan then easily be added to the marker compound mixture at anappropriate concentration for the type of product to be authenticated.In certain instances, the DNA taggant maybe mixed with other componentsof the marker compound without any prior encapsulation. Severalprocesses such as nucleic acid fragment encapsulation and othertechniques utilized for protecting nucleotides, and in particular, DNAfrom degradation, are well known in the art.

In other embodiments, the marker compound mixture is to be able tocamouflage or “hide” the specified nucleic acid tag with extraneous andnonspecific nucleic acid oligomers/fragments, thus making it difficultfor unauthorized individuals, such as forgers to identify the sequenceof the nucleic acid tag. In certain embodiments, the marker compoundcomprises a specified dsDNA taggant from a known source (i.e. mammal,invertebrate, plant and the like) along with genomic DNA from thecorresponding or similar DNA source. The amount of the DNA taggant foundin a marker compound varies depending on the particular product to beauthenticated, the duration the taggant needs to be viable (e.g. 1 day,1 month, 1 year, multiple years) prior to authentication, expectedenvironmental exposure, the detection method to be utilized, etc.

After the nucleic acid fragment/marker compound with a known nucleicacid sequence has been manufactured and applied to the item, the methodfurther comprises generating an item having a DNA fragment marker or tagin event 104. The particular product or item generated may be taggedwith a nucleic acid marker throughout the complete product or only in apredetermined region of the product. When the product to beauthenticated is a solid, a specified amount of nucleic acid markermaybe incorporated throughout the volume of the product, only on thesurface of the product or in some embodiments, placed only on apreviously designated section of the product.

In many embodiments the item to be tagged is an ink, paint or pigmentthat may be in liquid, powder or gel form. The nucleic acid marker ortaggant may be introduced to the ink at a desired concentration andintermixed with the ink. The ink may be present in a container orcartridge when the nucleic acid marker is added, or the labeled ink maybe subsequently transferred into printer cartridges, pens for signingdocuments, into official stamp ink pads or blotting pads such asutilized by a notary, spray containers, or other containers.

In certain embodiments the item generated is a printed item such as adocument or lithographic print. In such embodiments the nucleicacid-labeled ink may be applied to the document by various printtransfer techniques, or by brushing, spraying, blotting or other methodof applying ink to a document.

If the product is a textile garment, the marker could be either solid orliquid and applied to a predetermined area of the garment. Textiles mayhave a label with the manufactures name on it and may also be used as aregion of the product which the nucleic acid marker is placed. The aboveexamples are presented for clarity and are not meant to be limiting inscope.

In general, when the taggant is dsDNA, PCR is the technique for taggantdetection as described in event 110 below. The copy number of DNAtaggant in a predetermined sample size of marker compound used forauthentification is about 3 copies to about 100,000 copies, morepreferably about 10 copies to about 50,000 copies, and even morepreferably about 100 copies to about 10,000 copies of DNA taggant. Theconcentration of NA taggent within the ink or pigment may be varied asrequired depending upon particular embodiments of the invention. PCR caneffectively detect extremely small amounts of DNA taggant and skilledpersons can easily formulate DNA-labeled inks using the invention.

The embodiment of the method of authenticating and verifying an itemdepicted in FIG. 1 further comprises preparing the item to be verifiedas in step 106. In step 106, a sample may be collected of the particularitem of interest for verification, i.e., DNA analysis on whether theitem contains the nucleotide tag. For example, the preparation maycomprise sampling the ink or pigment within a printer cartridge or othercontainer. Where the item prepared is a document or printed item aportion of the document containing NA-tagged ink may be cut, scraped,abraded, or otherwise removed from the document for analysis.Preparation of the document may require cleaning or solvent treatmentprior to removing a sample portion of the document to be verified.Preparation of the item may occur without further purification, butusually, some extraction, isolation or purification of the nucleic acidtag obtained in the sample is required. Details on the extraction,concentration and purification techniques useful for the methods of theinvention are described more fully below and also in the examples.

In certain embodiments the placement or position of the NA marker on theitem of interest maybe located by the detection of materials orcompounds configured to or associated with the NA fragment in the NAmarker. Event 108 provides for such detection of the DNA marker. In manyembodiments the DNA marker may be bound or coupled to, or otherwiseassociated with, a chemically or optically detectable label. Detectionof DNA-labeled portions of the item may be carried out by opticallydetecting fluorescent dyes or upconverting phosphor particles which canbe detected easily by UV and/or IR portable light sources. Thus, forexample, a printed document could be examined with a UV or IR lightsource to find a particular region or regions of the document thatcontain a particular fluorescent marker. In this manner, only a smallportion of the item (as identified by the fluorescent dye or particles)needs to be sampled for DNA. The materials or compounds utilized forlocating the position of the NA marker on a document or item of interestmaybe coated with functional groups which can covalently bind to the NAfragment(s) of the NA marker, as described below. Event 108 may becarried out prior to event 106.

In general, analyzing the item for the presence of DNA in event 110,comprises providing a “detection molecule” configured to the nucleicacid tag. A detection molecule includes but is not limited to a nucleicacid probe and/or primer set which is complementary to the sequence ofthe nucleic acid taggant, or a dye label or color producing moleculeconfigured to bind and adhere to the nucleic acid taggant. When thedetection of the nucleic acid taggant comprises amplifying the nucleicacid taggant using PCR, the detection molecule(s) are primers whichspecifically bind to a certain sequence of the nucleic acid taggant.When real time PCR is utilized in the analysis of the sample, anidentifiable nucleotide probe may also be provided to enhance thedetection of the nucleic acid taggant as well as providesemi-quantitative or quantitative authentication results. With the useof real time PCR, results from the analysis of the sample can becompleted within 30 minutes to 2 hours, including extracting orpurifying the nucleic acid taggant from the collected sample. Variousembodiments utilize a wide range of detection methods besides for PCRand real time PCR, such as fluorescent probes, probes configured tomolecules which allow for the detection of the nucleic acid tag whenbound to the probe by Raman spectroscopy, Infrared spectroscopy or otherspectroscopic techniques used by those skilled in the art of nucleicacid detection.

The results of the analysis of the ink, ink cartridge, pigment, printeddocument or other item are reviewed to determine if the specific nucleicacid taggant is present in the sample. If so, in step 112, theauthentication of whether the item is genuine or not can be verified. Ifthe nucleic acid taggant is not found or detected in the item ofinterest, the conclusion from the analysis is that the item is notauthentic or has been tampered with as in step 116. If the nucleic acidtaggant is detected in the item, then the item is verified as beingauthentic as in step 114.

In some embodiments, the quantity or concentration of the nucleic acidtaggant within a collected sample can be determined and compared to theinitial amount of nucleic acid taggant placed in the product to allowfor the detection of fraud caused by diluting the product with inferiorproducts by forgers. In general, quantitative detection methods compriseproviding an internal or external control to evaluate the efficiency ofdetection from one sample/analysis to the next. The efficiency ofdetection may be affected by many parameters such as, probehybridization conditions, molecules or substances in the product whichmay interfere with detection, and/or primer integrity, enzyme quality,temperature variations for detection methods utilizing PCR. By providinga control, in the detection methods, any variable conditions can benormalized to obtain an accurate final concentration of the nucleic acidtag in the product.

Incorporation of Functional Groups

In certain embodiments, the nucleic acid tag is labeled with at leastone compound or “detection molecule” prior to being incorporated intothe specified product to aid in the extraction and/or detection (seeevent 108 above) of the nucleic acid marker from the product after beingplaced in a supply chain. A detection molecule is a molecule or compoundwith at least one functionality. For example, fluorescent molecules,which may be in particulate form, may be configured to the nucleic acidmarker for certain detection methods which are described in detailbelow.

In certain preferred aspects, suitable dyes include, but are not limitedto, coumarin dyes, xanthene dyes, resorufins, cyanine dyes,difluoroboradiazaindacene dyes (BODIPY), ALEXA dyes, indoles, bimanes,isoindoles, dansyl dyes, naphthalimides, phthalimides, xanthenes,lanthanide dyes, rhodamines and fluoresceins. In other embodiments,certain visible and near Infrared (IR) dyes and IR materials are knownto be sufficiently fluorescent and photostable to be detected as singlemolecules. In this aspect the visible dye, BODIPY R6G (525/545), and alarger dye, LI-COR's near-infrared dye, IRD-38 (780/810) can be detectedwith single-molecule sensitivity and are used to practice theauthentication process described herein. In certain embodiments,suitable dyes include, but are not limited to, fluorescein,5-carboxyfluorescein (FAM), rhodamine,5-(2′-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS),anthranilamide, coumarin, terbium chelate derivatives, Reactive Red 4,BODIPY dyes and cyanine dyes.

There are many linking moieties and methodologies for attachingfluorophore or visible dye moieties to nucleotides, as exemplified bythe following references: Eckstein, editor, Oligonucleotides andAnalogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckerman etal., Nucleic Acids Research, 15: 5305-5321 (1987) (3′ thiol group onoligonucleotide); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′ sulfhydryl); Giusti et al., PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′ phosphoamino groupvia Aminolink™ II available from Applied Biosystems, Foster City,Calif.) Stabinsky, U.S. Pat. No. 4,739,044 (3′ aminoalkylphosphorylgroup); AP3 Labeling Technology (U.S. Pat. Nos. 5,047,519 and 5,151,507,assigned to E.I. DuPont de Nemours & Co); Agrawal et al, TetrahedronLetters, 31: 1543-1546 (1990) (attachment via phosphoramidate linkages);Sproat et al., Nucleic Acids Research, 15: 4837 (1987) (5′ mercaptogroup); Nelson et al, Nucleic Acids Research, 17: 7187-7194 (1989) (3′amino group); and the like.

In other embodiments, a nucleic acid probe complementary to the nucleicacid marker is labeled with at least one compound or molecule withfunctionality to aid in the detection of the nucleic acid tag/marker.The techniques and dyes utilized in labeling the nucleic acid tag or thecomplementary probe are the same due to the nucleic acid nature of thetag and probe.

The detection molecules of the invention can be incorporated into probemotifs, such as Taqman probes (Held et al., Genome Res. 6: 986-994(1996), Holland et al., Proc. Nat. Acad. Sci. USA 88: 7276-7280 (1991),Lee et al., Nucleic Acids Res. 21: 3761-3766 (1993)), molecular beacons;Tyagi et al., Nature Biotechnol., 16:49-53 (1998), U.S. Pat. No.5,989,823, issued Nov. 23, 1999)) scorpion probes (Whitcomb et al.,Nature Biotechnology 17: 804-807 (1999)), sunrise probes (Nazarenko etal., Nucleic Acids Res. 25: 2516-2521 (1997)), conformationally assistedprobes (Cook, R., copending and commonly assigned U.S. ProvisionalApplication No. 60/138,376, filed Jun. 9, 1999), peptide nucleic acid(PNA)-based light up probes (Kubista et al., WO 97/45539, December1997), double-strand specific DNA dyes (Higuchi et al, Bio/Technology10: 413-417 (1992), Wittwer et al, Bio/Techniques 22: 130-138 (1997))and the like. These and other probe motifs with which the presentdetection molecules can be used are reviewed in Nonisotopic DNA ProbeTechniques, Academic Press, Inc. 1992.

In other embodiments, the molecular beacon system is utilized to detectand quantify the nucleic acid tag from the product of interest.“molecular beacons” are hairpin-shaped nucleic acid detection probesthat undergo a conformational transition when they bind to their targetthat enables the molecular beacons to be detected. In general, the loopportion of a molecular beacon is a probe nucleic acid sequence which iscomplementary to the nucleic acid marker. The stem portion of themolecular beacon is formed by the annealing of arm sequences of themolecular beacon that are present on either side of the probe sequence.A functional group such as a fluorophore (e.g. coumarin, EDNAS,fluorescein, lucifer yellow, tetramethylrhodamine, texas red and thelike) is covalently attached to the end of one arm and a quenchermolecule such as a nonfluorescent quencher (e.g. DABCYL) is covalentlyattaches to the end of the other arm. When there is no target (nucleicacid tag) present, the stem of the molecular beacon keeps the functionalgroup quenched due to its close proximity to the quencher molecule.However, when the molecular beacon binds to their specified target, aconformational change occurs to the molecular beacon such that the stemand loop structure cannot be formed, thus increasing the distancebetween the functional group and the quencher which enables the presenceof the target to be detected. When the functional group is afluorophore, the binding of the molecular beacon to the nucleic acid tagis detected by fluorescence spectroscopy.

In certain embodiments, a plurality of nucleic acid tags with varyingsequences are used in labeling a particular product. The differentnucleic acid tags can be detected quantitatively by a plurality ofmolecular beacons, each with a different colored fluorophore and with aunique probe sequence complementary to at least one of the plurality ofnucleic acid tags. Being able to quantitate the various fluorphores(i.e. various nucleic acid tags) provides a higher level ofauthentication and security. It should be noted, that the otherfunctional groups described above useful in labeling nucleic acid probescan also be utilized in molecular beacons for the present invention.

In other embodiments of the invention, the methods for authenticating anitem comprise labeling the item with an optical reporter marker linkedto a nucleic acid tag, detecting the optical reporter, and thencharacterizing or verifying the nucleic acid taggant associated with theitem in an effective manner, by nucleic acid sequencing, genotyping orlike techniques. This embodiment allows for verification of tagged itemsin a manner that's helps prevent forgers counterfeit producers fromsubstituting false or counterfeit goods in place of authentic items.

FIG. 2 is a flow chart illustrating generally a method 200 forauthenticating an item with a nucleic acid-linked optical reportermarker in accordance with the invention. The method 200 comprises, atevent 210, providing an optical reporter marker having a nucleic acidtaggant linked to an optical reporter particle, the nucleic acid tagganthaving a known portion of its sequence identifiable or sequenceable.

The optical reporter particle of event 210 is a light emitting opticalreporter and in most embodiments is an upconverting phosphor particle(UCP). In certain embodiments the upconverting phosphor particle UCP iscoated with a silylination composition which is configured to covalentlylink to the nucleic acid taggant. Specific UCPs usable with theinvention are described further below.

The nucleic acid (NA) taggant of event 210 may be DNA, cDNA, or anyother nucleic acid fragment comprising nucleic acids or nucleic acidderivatives. The NA maybe a nucleic acid fragment that is singlestranded or preferably double stranded and may vary in length, dependingon the item to be labeled as well as the detection technique utilized inthe nucleic acid detection process.

The nucleic acid marker may be synthetically produced using a nucleicacid synthesizer or by isolating nucleic acid material from yeast, humancell lines, bacteria, animals, plants and the like. In certainembodiments, the nucleic acid material may be treated with restrictionenzymes and then purified to produce an acceptable nucleic acidmarker(s). The length of the nucleic acid tag usually ranges betweenabout 50 to about 1 kilo bases, more usually about 100 bases to about800 bases, and preferably 150 bases to about 500 b in length.

The nucleic acid taggant may comprise one specific nucleic acid sequenceor alternatively, may comprise a plurality of various nucleic acidsequences. In one embodiment, polymorphic DNA fragments of the typeshort tandem repeats (STR) or single nucleotide polymorphisms (SNP) areutilized as an anti-counterfeit nucleic acid tag. While the use of asingle sequence for a nucleic acid marker may make detection of themarker easier and quicker, the use of a plurality of nucleic acidsequences such as STR and SNP, in general, give a higher degree ofsecurity against forgers.

In certain embodiments of the methods of the invention, the nucleic acidtaggant is derived from DNA extracted from a specific plant source andis specifically digested and ligated to generate artificial nucleic acidsequences which are unique to the world. The digestion and ligation ofthe extracted DNA is completed by standard restriction digestion andligase techniques known to those skilled in the art of molecularbiology.

The optical reporter marker compound may be produced as a solid orliquid, water or oil based, a suspension, an aggregate or the like. Theoptical reporter marker allows for easy detection of where the opticalreporter marker is located on or within the item of interest with basichigh intensity light emitting equipment such as a hand-held ultraviolet(UV) lamp, IR emitting diode, hand-held IR laser and the like.

The optical reporter marker also enables the authentication of the itemor ink of interest by both confirming that the correct emissionspectra/wavelength for the optical reporter particle is detected as wellas being able to locate and determine by sequencing if the nucleic acidtaggant comprises the correct nucleic acid sequence.

In certain embodiments, the optical reporter marker may camouflage or“hide” a specified nucleic acid tag of verifiable sequence by includingextraneous and nonspecific nucleic acid oligomers/fragments, thus makingit difficult for unauthorized individuals such as forgers to identifythe sequence of the nucleic acid tag. In certain embodiments, theoptical reporter marker comprises a specified dsDNA taggant from a knownsource (i.e. mammal, invertebrate, plant and the like) along withgenomic DNA from the corresponding or similar DNA source. The amount ofthe DNA taggant found in a optical reporter marker compound may varydepending on the item to be authenticated, the duration or shelf-lifethe taggant needs to be viable (e.g. 1 day, 1 month, 1 year, multipleyears) prior to authentication, expected environmental exposure, thedetection method to be utilized, and other factors.

The method 200 for authenticating an item further comprises, in event120, applying or introducing the nucleic acid-linked optical reportermarker to an item of interest in event. The nucleic acid-linked opticalreporter marker may be applied in a specific, pre-determined amount orquantity. The item may be labeled with an optical reporter markerthroughout the complete item, as a coating over the entire item, or onlyin a predetermined region or portion of the item. The marker may beapplied in liquid solution, liquid dispersion, paste, powder, or otherform. Application of the marker may be carried out using an eye-dropper,spoon, spatula, syringe, or other applicator tool. When the item to beauthenticated is a solid, a specified amount of optical reporter markermaybe incorporated throughout the volume of the item, or only on thesurface of the item or, in some embodiments, placed only on a previouslydesignated section or portion of the item. In embodiments where the itemto be authenticated is a fungible powder, the nucleic acid-lined opticalreporter may be dispersed throughout the powdered material.

If the item is a textile or garment item, the marker could be eithersolid or liquid form of ink and applied to a predetermined area of thegarment. Textiles may have a label with the manufactures name on it andmay also be used as a region of the garment which the optical reportermarker is placed. The marker may be introduced, for example, by applyinga liquid solution or suspension of the marker onto a selected portion ofthe garment and allowing the solution or suspension to dry by solventevaporation to leave the markers in place. The marker can also beintroduced by applying a binding solution containing DNA marker to thegarment.

In embodiments where item to be authenticated is an ink, paint orpigment that may be in liquid, powder or gel form, the nucleic acidlabeled optical reporter may be introduced to the ink at a desiredconcentration and intermixed with the ink as noted above. The ink may bepresent in a container or cartridge when the nucleic acid marker isadded, or the labeled ink may be subsequently transferred into printercartridges, pens for signing documents, into official stamp ink pads orblotting pads such as utilized by a notary, spray containers, or othercontainers. Where the item to be authenticated is a printed item such asa document or lithographic print, the nucleic acid-labeled ink may beapplied to the document by various print transfer techniques, or bybrushing, spraying, blotting or other method of applying ink to adocument.

The authentication method 200 further comprises, in event 230, detectingthe nucleic acid-linked optical reporter tag associated with the item ofinterest. Usually the detecting of the optical reporter markerassociated with the item occurs after a period of time has lapsed. Forexample, after tagging the marked item may be introduced into a supplychain or the item may be placed into service. Frequently, forgers havethe best access to items when they are being shipped from themanufacturer/producer to a retail outlet or location. Forgers also haveaccess to the items of interest during maintenance or service of certainof products, such as aircraft, where the item of interest is inspectedor replaced (i.e. fasteners). Having a method in which the producer cantrack and authenticate items or goods allows for a better monitoring ofwhen and where counterfeit goods are being replaced with forgeries orotherwise being tampered with.

Detecting the optical reporter particle(s) represents a first level ofauthentication of the item. When the optical reporter particle is anupconverting phosphor particle, the marker can be detected by a highenergy invisible light source such as an infrared laser, which may behand-held and manipulated by a user, or suitably mounted to allow goodsto be positioned in the lamp output. The infrared light is absorbed bythe optical reporter particles, which in turn emit light at a wavelengththat is characteristic of the optical reporter particle. Variousupconverting phosphor compositions that provide selectable outputwavelengths are known in the art, as described further below, and may beused with the invention. Once the optical reporter has been locatedwithin or on the item of interest, obtaining a sample of the opticalreporter marker may occur at event 240.

In event 240, a sample is collected from the item of interest having theoptical reporter marker. In certain embodiments, this may comprisevisually inspecting the marker compound found in event 230, and/orscraping, cutting or dissolving a portion of the marked item to obtain asample for analysis. When the item has entered a supply chain or hasbeen in service, a manufacturer or an authorized individual can collecta sample of the optical reporter marker from the item at any desiredpoint along the supply chain or during the service or routinemaintenance of an item where the item is utilized for authenticationpurposes. The collecting of the sample may be carried out, for example,by wiping the item with a cloth (which may be moistened with solvent) toremove the marker from the item. The sample collecting in otherembodiments may be achieved using a cutting, gouging, scraping,abrading, or other sampling tool configured to remove a portion of theitem containing the optical reporter marker.

The embodiment of FIG. 2 further comprises analyzing the collectedsample for the presence of the nucleic acid taggant in event 250. Inmany embodiments the analyzing of the collected sample comprisesdetermining the DNA sequence of the nucleic acid taggant, and comparingthe determined DNA sequence with a known or reference DNA sequence. Theanalysis of the sample collected from the item may occur without furtherpurification, but in many embodiments some form of extraction, isolationor purification of the nucleic acid tag obtained in the sample may berequired. Details on the extraction, concentration and purificationtechniques useful for the methods of the invention are described morefully below and also in the examples.

In general, analyzing the sample comprises providing a “detectionmolecule” configured to the nucleic acid tag. A detection moleculeincludes but is not limited to a nucleic acid probe and/or primer setwhich is complementary to at least a portion of the sequence of thenucleic acid taggant, or a dye label or color-producing moleculeconfigured to bind and adhere to the nucleic acid taggant. The detectionof the nucleic acid taggant may further comprise amplifying the nucleicacid taggant using PCR, with the detection molecule(s) being primerswhich specifically bind to a certain sequence of the nucleic acidtaggant. When real time PCR is utilized in the analysis of the sample,an identifiable nucleotide probe may also be provided to enhance thedetection of the nucleic acid taggant as well as providesemi-quantitative or fully quantitative authentication results. With theuse of real time PCR, results from the analysis of the sample can becompleted within 30 minutes to two hours, including extracting orpurifying the nucleic acid taggant from the collected sample. Variousembodiments of the invention may utilize a wide range of detectionmethods besides for PCR and real time PCR, such as DNA microarray,fluorescent probes, probes configured to molecules which allow for thedetection of the nucleic acid tag when bound to the probe by Ramanspectroscopy, Infrared spectroscopy or other spectroscopic techniquesused by those skilled in the art of nucleic acid detection. The methodutilized to detect the nucleic acid is dependent on the quantity ofnucleic acid taggant associated with the optical reporter marker. Whenonly a few copies of NA taggant are collected in the marker sample, highsensitivity techniques such as PCR maybe preferable over fluorescentprobes.

In event 260 the results of the analysis of the collected sample arereviewed and a query or determination is made as to whether or not thespecific nucleic acid taggant was detected in the sample. If the nucleicacid taggant is not found or not detected in the collected sample of theitem of interest at event 260, the conclusion at event 270 from theanalysis is the that item is not authentic or has been tampered with. Ifthe nucleic acid taggant is detected in the sample at event 260, thenthe item is verified in event 280 as being authentic.

If a determination is made in event 270 that an item is not authentic, adifferent, earlier point in the supply or commerce chain may be selectedand events 230 through 260 may be repeated. Thus an item from an earlierpoint in the supply chain would be selected, the optical reporter markerdetected, and a sample collected and analyzed. If it is again determinedthat the item is not authentic or has been otherwise tampered with, thenevents 230-260 may be repeated with an item selected from yet an earlierpoint in the supply chain. In this manner, the time and/or location oftampering or counterfeit substitute may be located.

In some embodiments, the quantity or concentration of the nucleic acidtaggant within a collected sample can be determined and compared to theinitial amount of nucleic acid taggant placed in the item to allow forthe detection of fraud caused by diluting the item with inferiorproducts by forgers. In general, such quantitative detection wouldfurther comprise, in event 250, providing an internal or externalcontrol to evaluate the efficiency of detection from one sample/analysisto the next. The efficiency of detection may be affected by manyparameters such as, probe hybridization conditions, molecules orsubstances in the good which may interfere with detection, and/or primerintegrity, enzyme quality, temperature variations for detection methodsutilizing PCR. By providing a control, in the detection methods, anyvariable conditions can be normalized to obtain an accurate finalconcentration of the nucleic acid taggant in the item.

In certain embodiments a plurality of nucleic acid tags with varyingsequences associated with a corresponding plurality of optical reportersmay be used in labeling a single item. The different nucleic acid tagscan be detected qualitatively by the plurality of optical reporters,each with a different emission wavelength linked to a uniquesequenceable nucleic acid taggant.

Compounds Utilized in the Methods of the Invention

The methods of authentification of an item of the invention comprisecompounds of the formula I:

(cOpR)-[L-(NA)]_(m)

wherein:

m is an integer greater than 1;

(cOpR) is a coated optical reporter particle;

(NA) is a nucleic acid oligomer of detectable sequence; and

L is a linking group covalently bound to the coated optical reporterparticle and to the nucleic acid oligomer.

While formula I specifically relates to linking nucleic acid oligomersor nucleotides to the surface of the coated optical reporter particle,it should be understood to the those skilled in the art that otherbiomolecules besides nucleotides can be covalently linked to L. Suchbiomolecules include but are not limited to peptides, proteins,antibodies, enzymes, DNA binding proteins and the like. Thesebiomolecules, maybe modified to include lipids, carbohydrates,fluorescent and/or upconverting phosphor molecules or other detectablecompounds or markers.

In many embodiments, NA is a DNA oligomer. The DNA oligomer maybe eithersingle stranded DNA or double stranded DNA. In certain embodiments NAmaybe comprise cDNA, RNA, STR (single tandem repeat) or SNP (singlenucleotide polymorphism). NA oligomers of the compositions of theinvention may also be modified to comprise at least one dUTP nucleicacid or at least one nucleic acid within the oligomer which has beenmodified to contain a detectable marker.

In many embodiments NA is a DNA oligomer having a length of betweenabout 40 base pairs and about 1000 base pairs (per strand).

In other embodiments the DNA has a length of between about 80 and 500base pairs (per strand).

In yet other embodiments the DNA has a length of between about 100 toabout 250 base pairs (per strand).

The DNA used with the invention maybe natural or synthetically produced.All or a portion of the DNA may comprise an identifiable sequence.

In certain embodiments of formula I, the coated optical reportercomprises a visible or infrared detectable light emitting materialselected from the group consisting of a fluorescent dye, an upconvertingphosphor, a ceramic powder, or a quantum dot material. In mostembodiments where the cOpR comprises a visible or infrared detectablelight emitting material, the light emitting materials are excitable byUV, visible or an infrared light source.

In some embodiments, rare earth-doped ceramic particles are used asphosphor particles. Phosphor particles may be detected by any suitablemethod, including but not limited to up-converting phosphor technology(UPT), in which up-converting phosphors transfer lower energy infrared(IR) radiation into higher-energy visible light. Although anunderstanding of the mechanism is not necessary to practice the presentinvention and the present invention is not limited to any particularmechanism of action, in some embodiments the UPT up-converts infraredlight to visible light by multi-photon absorption and subsequentemission of dopant-dependant phosphorescence (See, e.g., U.S. Pat. No.6,399,397; van De Rijke, et al., Nature Biotechnol. 19(3):273-6 (2001);Corstjens, et al., IEE Proc. Nanobiotechnol. 152(2):64 (2005), eachincorporated by reference herein in its entirety.

In many embodiments, the phosphor nanoparticles utilized in the methodsof the invention may be of the formula A

(Y_(x)RE¹ _(y), RE² _(z))₂O₃  A

wherein:

RE¹ and RE² each is a different rare earth element;

x is from about 0.6 to about 0.95;

y is from 0 to about 0.35; and

z is from 0 0.1 to about 0.001;

provided that y and z are not simultaneously equal to 0.

The rare earth elements RE¹ and RE² may each independently be selectedfrom Ytterbium, Erbium, Holmium, Thulium, or Terbium.

In many embodiments RE¹ is Ytterbium.

In many embodiments RE² is Erbium.

The up-converting particles utilized in the methods of the invention maybe spherical, non-agglomerated, non-porous particles with an averagesize of 40-60 nm. In general, particle sizes may range from about 10 nmto about 5 um in size. Such up-converting phosphor nanopowders such asdoped yttrium oxide and yttrium oxysulfide are commercially availableand may be obtained from such as Nanocerox, Inc., of Ann Arbor, Mich.

Suitable examples of up-converting phosphors are compounds of rareearths or elements from the group IIIB such as: Na-yttrium fluoride,lanthanum fluoride, lanthanum oxysulphide, yttrium oxysulphide, yttriumfluoride, yttrium gallate, gadolinium fluoride, barium-yttriumfluorides, gadolinium oxysulphide as well as compounds of the above typedoped with activator pairs such as ytterbium/erbium, ytterbium/thuliumor ytterbium/holmium. Other suitable up-converting phosphors includechelate compounds of erbium, neodymium, thulium, holmium andpraseodymium.

The following compositions are merely illustrative of some of theup-converting phosphor containing compositions that can be synthesizedby the synthetic reaction schemes of the methods of the presentinvention. Various modifications to these synthetic reaction schemes canbe made and will be suggested to one skilled in the art having referredto the disclosure contained in this Application.

TABLE I Upconverting Phosphor Compositions Phosphor Material AbsorberIon Emitter Ion Oxysulfides (O₂S) Y₂O₂S Ytterbium Erbium Gd₂O₂SYtterbium Erbium La₂O₂S Ytterbium Holmium Oxyhalides (OX_(y)) YOFYtterbium Thulium Y₃OCl₇ Ytterbium Terbium Fluorides (F_(x)) YF₃Ytterbium GdF₃ Ytterbium Erbium LaF₃ Ytterbium Erbium NaYF₃ YtterbiumHolmium BaYF₅ Ytterbium Thulium BaY₂F₈ Ytterbium Thulium Gallates(Ga_(x)O_(y)) YGaO₃ Ytterbium Erbium Y₃Ga₅O₁₂ Ytterbium Erbium Silicates(Si_(x)O_(y)) YSi₂O₅ Ytterbium Holmium YSi₃O₇ Ytterbium Thulium

In certain embodiments the coated optical reporter used in the methodsof the invention may also comprise at least one electromagnetic emittingmaterial. An electromagnetic emitting material as part of thecomposition of the invention, allows for the composition to be detectedby various methods and devices. Where the electromagnetic emittingmaterial is detectable by mechanical devices which provide at least onesource selected from the group consisting of an infrared radiationsource, magnetic field source or electromagnetic pulse. Thiselectromagnetic emitting material may be in conjunction with at leastone light emitting material, such as an upconverting phosphor.

When the compositions used in the methods of authenticating an item ofthe invention comprise UCPs, the upconverting phosphor material/particlein certain embodiments have the formula B

Y_(x)Yb_(y)Er_(z)O₂S  B

wherein:

x is from about 0.6 to about 0.95;

y is from about 0.05 to about 0.35; and

z is from about 0.1 to about 0.001.

In other embodiments, the upconverting phosphor particle may be of theformula C:

Na(Y_(x)Yb_(y)Er_(z))F₄  C

wherein

x is from about 0.6 to about 0.95

y is from about 0.05 to about 0.35; and

z is from about 0.1 to about 0.001.

In certain embodiments of formula I, L comprises an alkylene moietyhaving a first end covalently bound to the coated optical reporterparticle (cOpR) and a second end covalently bound to the nucleic acidoligomer (NA).

In many embodiments of formula I, L is of the formula D:

-A-R¹—B—  D

wherein:

R¹ is C₂₋₈alkylene;

-A- is a group covalently bonded to the surface of the coated opticalreporter; and

—B— is a group covalently bonded to the 3′ or 5′ end of the nucleic acidoligomer.

In certain embodiments of formula D, —R¹— is —(CH₂)_(n)— and n is from 2to 8.

In certain embodiments of formula D, —B— is:

—S—;

—O—;

—NR^(a)—;

—S—(CH₂)_(p)—;

—O—(CH₂)_(p)—;

—NR^(a)—(CH₂)_(p)—;

—S—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—;

—O—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—,

—NR^(a)—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—;

—S—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—;

—O—C(O)—(CH₂)_(r)C(O)—NR —(CH₂)_(p)—; or

—NR^(a)—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—;

wherein:

p is from 2 to 8;

q is from 1 to 8;

r is from 2 to 8; and

each R^(a) is independently hydrogen or a C₁₋₆alkyl.

In certain embodiments of formula D, —B— is:

—S—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p) or

—NR^(a)—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—;

wherein:

p is from 2 to 8;

q is from 1 to 8;

r is from 2 to 8; and

each R^(a) is independently hydrogen or a C₁₋₆alkyl.

In other embodiments of formula D, —B— is:

—S—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p) or

—NR^(a)C(O)—(CH₂)_(r)—C(O)—NR²—(CH₂)_(p)—;

wherein:

p is from 2 to 6;

q is from 1 to 3; and

r is 2 or 3.

In other embodiments of formula D, —B— is

—S—CH₂—C(O)—NH—(CH₂)₆—

or

—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₆—.

In certain embodiments of formula D, -A- is —O—.

In many embodiments of formula I, the coated optical reporter (cOpR) iscoated with silica. Usually when the coated optical reporter comprises acoating of silica, the silica comprises at least one Si—O bond.

The value of m in formula I will vary according to the surface area ofthe coated optical reporter and the number of functional groups on theoptical reporter surface cable of bonding to -L-. The value of m isalways greater than one, and usually greater than 10. Preferably m isgreater than 100, and in many embodiments m is greater than 10³. In manyembodiments m may be, for example, between about 10 and about 10⁹. Incertain embodiments m may be from about 100 to about 10⁸. In someembodiments m may be from about 10³ to about 10⁷.

In certain embodiments the compositions used in the methods of theinvention are of the formula II:

(UCP)—[A-R¹—X—R²—C(O)—NR^(a)—R³-(DNA)]_(m)  II

wherein:

m is an integer greater than 1;

UCP is an upconverting phosphor particle;

DNA is a single or double stranded deoxy-ribonucleic acid oligomer;

-A- is a group capable of covalently bonding to the surface of theUpconverting phosphor particle;

R¹ is C₂₋₈alkylene,

R² is C₁₋₈alkylene or —C(O)—C₁₋₈alkylene-;

—X— is —O—, —S— or —NR^(a)—;

R³ is C₂₋₈alkylene; and

R^(a) is hydrogen or C₁₋₆alkyl.

In certain embodiments of the invention, the subject composition may beof formula III:

(UCP)-[O—R¹—X—R²—C(O)—NH—R³-DNA]_(m)  III

wherein m, R¹, R², R³, UCP and DNA are as defined herein.

In certain embodiments of the invention, R¹ is C₂₋₆alkylene.

In certain embodiments of the invention, R² is C₁₋₆alkylene.

In certain embodiments of the invention, R³ is C₂₋₆alkylene.

In certain embodiments of the invention, R² is —C(O)—C₂₋₆alkylene-.

In certain embodiments of the invention, R¹ is —(CH₂)_(s)— wherein s isfrom 2 to 6. In some embodiments s is 3.

In certain embodiments of the invention, R² is —(CH₂)_(t)— wherein t isfrom 1 to 6. In some embodiments t is 1.

In certain embodiments of the invention, R² is —C(O)—(CH₂)_(u)— whereinu is from 1 to 6. In some embodiments u is 2 or 3, preferably 2.

In certain embodiments of the invention, R³ is —(CH₂)_(v)— wherein v isfrom 2 to 6. In some embodiments v is 6.

In certain embodiments of the invention, the subject composition may beof formula IV:

(UCP)-[O—(CH₂)_(s)—S—(CH₂)_(t)—C(O)—NH—(CH₂)_(v)-(DNA)]_(m)  IV

wherein:

s is from 2 to 6;

v is from 2 to 6;

t is from 1 to 3; and

m, UCP and DNA are as defined herein.

In certain embodiments of the invention, the compositions may be offormula V:

(UCP)-[O—(CH₂)_(s)—NH—C(O)—(CH₂)_(u)—C(O)—NH—(CH₂)_(v)-(DNA)]_(m)  V

wherein:

s is from 2 to 6;

v is from 2 to 6;

u is 2 or 3; and

m, UCP and DNA are as defined herein.

In certain embodiments of the invention, the compositions may be offormula VI:

(UCP)-[O—(CH₂)₃—S—CH₂—C(O)—NH—(CH₂)₆-(DNA)]_(m)  VI

wherein m, UCP and DNA are as defined herein.

In certain embodiments of the invention, the compositions may be offormula VII:

(UCP)-[O—(CH₂)₃—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₆-(DNA)]_(m)  VII

wherein m, UCP and DNA are as defined herein.

Encapsulation of a Nucleic Acid Tag

In some embodiments, the nucleic acid marker is incorporated into theproduct in the presence of molecules which encapsulate the nucleic acidmarker by forming microspheres. Encapsulating the nucleic acid markerhas the benefit of preventing the nucleic acid marker from degradingwhile present in a supply chain or during the use of the marked product.The encapsulating materials in most embodiments are of plant origin butmay also be synthetically produced materials. The encapsulation of anucleic acid tag comprises placing the nucleic acid tag into a solventwith a polymer configured to form a microshpere around the tag. Thepolymers used can be selected from biodegradable or non-biodegradablepolymers. Preferred biodegradable polymers are those such as lactic andglycolic acids and esters such as polyanhydrides, polyurethantes,butryic polyacid, valeric polyacid, and the like. Non biodegradablepolymers appropriate for encapsulation are vinyletylenene acetate andacrylic polyacid, polyamides and copolymers as a mixture thereof. Thepolymers can also be selected from natural compounds such as dextran,cellulose, collagen, albumin, casein and the like.

Certain aspects of the invention comprise labeling the microspheres tobenefit in the capture of the nucleic acid tag during the extraction ofthe label from the product of interest. The microspheres may comprisemagnetically charged molecules which allow the microspheres containingthe nucleic acid tag to be pulled out of a solution by a magnet.

The microspheres can also be labeled with streptavidin, avidin,biotinylated compounds and the like. Labeling the microspheres aids inthe purification of the nucleic acid tag prior to detection and also isuseful in concentrating the nucleic acid tag so as to enable in someembodiments, the nucleic acid tag to be detected without PCRamplification.

In other embodiments, the nucleic acid marker is applied or added to theproduct without being encapsulated in microspheres. For example, thenucleic acid marker may be dissolved in a solution compatible with thecomposition of the particular product such as a textile and then thesolution comprising the nucleic acid marker is placed on the surface ofthe textile product, allowing the nucleic acid marker to be absorbedinto the fabric.

Incorporation of the Nucleic Acid Tag into the Particular Item ofInterest

The method of incorporating the nucleic acid tag into an item ofinterest depends significantly on the type of product to beauthenticated as described above. The nucleic acid tag maybe added to amarker compound in a “naked” or encapsulated form at a predetermineconcentration which allows for accurate detection of the nucleic acidtaggant. The marker compound is generally a liquid but in certainembodiments is a solid. The marker compound maybe a liquid and after theaddition of the nucleic acid taggant, is dried prior to introducing themarker as an inert substance of a particular product. When the markercompound comprising a nucleic acid taggant is in liquid form, the markercompound is generally applied to the product in a lacquer, paint orliquid aerosol form.

In other embodiments the nucleic acid taggant may be applied to thefinished document as a paint/ink on a pre-designated position on thedocument. The ink utilized is formulated to allow detection of an upconverting phosphor particle, with minimal quenching of the lightemission from the UCP when excited by the appropriate light source.

When the document is a painting, for example, the nucleic acid taggantcan be mixed with paints appropriate for the type of painting beingmarked. The NA taggant is added to the paint mixture at an appropriateconcentration to allow for adequate detection of the NA marker. If theNA taggant marker comprises an UCP composition, the paint mixture iscompatible with the NA taggant as to not quench the emission of the UCPparticle. In some instances, the NA taggant marker may be introduced tothe painting as a topcoat or varnish as a topical application on thepainting.

Nucleic Acid Tag Extraction and Capture Methods

A variety of nucleic acid extraction solutions have been developed overthe years for extracting nucleic acid sequences from a sample ofinterest. See, for example, Sambrook et al. (Eds.) Molecular Cloning,(1989) Cold Spring Harbor Press. Many such methods typically require oneor more steps of, for example, a detergent-mediated step, a proteinasetreatment step, a phenol and/or chloroform extraction step, and/or analcohol precipitation step. Some nucleic acid extraction solutions maycomprise an ethylene glycol-type reagent or an ethylene glycolderivative to increase the efficiency of nucleic acid extraction whileother methods only use grinding and/or boiling the sample in water.Other methods, including solvent-based systems and sonication, couldalso be utilized in conjunction with other extraction methods.

In some embodiments, the authentication process comprises capturing thenucleic acid tag directly with a complementary hybridization probeattached to a solid support. In general, the methods for capturing thenucleic acid tag involve a material in a solid-phase interacting withreagents in the liquid phase. In certain aspects, the nucleic acid probeis attached to the solid phase. The nucleic acid probe can be in thesolid phase such as immobilized on a solid support, through any one of avariety of well-known covalent linkages or non-covalent interactions. Incertain aspects, the support is comprised of insoluble materials, suchas controlled pore glass, a glass plate or slide, polystyrene,acrylamide gel and activated dextran. In other aspects, the support hasa rigid or semi-rigid character, and can be any shape, e.g. spherical,as in beads, rectangular, irregular particles, gels, microspheres, orsubstantially flat support. In some embodiments, it can be desirable tocreate an array of physically separate sequencing regions on the supportwith, for example, wells, raised regions, dimples, pins, trenches, rods,pins, inner or outer walls of cylinders, and the like. Other suitablesupport materials include, but are not limited to, agarose,polyacrylamide, polystyrene, polyacrylate, hydroxethylmethacrylate,polyamide, polyethylene, polyethyleneoxy, or copolymers and grafts ofsuch. Other embodiments of solid-supports include small particles,non-porous surfaces, addressable arrays, vectors, plasmids, orpolynucleotide-immobilizing media.

As used in the methods of capturing the nucleic acid tag, a nucleic acidprobe can be attached to the solid support by covalent bonds, or otheraffinity interactions, to chemically reactive functionality on thesolid-supports. The nucleic acid can be attached to solid-supports attheir 3′, 5′, sugar, or nucleobase sites. In certain embodiments, the 3′site for attachment via a linker to the support is preferred due to themany options available for stable or selectively cleavable linkers.Immobilization is preferably accomplished by a covalent linkage betweenthe support and the nucleic acid. The linkage unit, or linker, isdesigned to be stable and facilitate accessibility of the immobilizednucleic acid to its sequence complement. Alternatively, non-covalentlinkages such as between biotin and avidin or streptavidin are useful.Examples of other functional group linkers include ester, amide,carbamate, urea, sulfonate, ether, and thioester. A 5′ or 3′biotinylated nucleotide can be immobilized on avidin or streptavidinbound to a support such as glass.

Depending on the initial concentration of the nucleic acid tag added tothe product of interest, the tag can be detected quantitatively withoutbeing amplified by PCR. In some embodiments, a single stranded DNA taglabeled with a detection molecule (i.e. fluorophore, biotin, etc.) canbe hybridized to a complementary probe attached to a solid support toallow for the specific detection of the “detection molecule” configuredto the tag. The nucleic acid DNA tag can also be double stranded, withat least one strand being labeled with a detection molecule. With adsDNA tag, the nucleic acid tag must be heated sufficiently and thenquick cooled to produce single stranded DNA, where at least one of thestrands configured with a detection molecule is capable of hybridizingto the complementary DNA probe under appropriate hybridizationconditions.

In certain aspects of the invention, the complementary probe is labeledwith a detection molecule and allowed to hybridize to a strand of thenucleic acid tag. The hybridization of the probe can be completed withinthe product, when the product is a textile or can be completed after thenucleic acid tag/marker has been extracted from the product, such aswhen the products are liquid (e.g. oil, gasoline, perfume, etc.). Thedirect detection methods described herein depend on having a largeinitial concentration of nucleic acid label embedded into the product orrigorous extraction/capture methods which concentrate the nucleic acidtag extracted from a large volume or mass of a particular product.

In one embodiment, where the NA taggant comprises an up convertingparticle, the extraction of the NA taggant marker varies depending on ifthe document being authenticated. when the NA marker comprises a UCPparticle, the NA marker can be located by detecting the presence of theUCP by an appropriate light source. The NA marker can then be extractedfrom the document by scraping, cutting out, or dissolving the portion ofthe document which is determined to have the presence of the correctup-converting phosphor particle(s). Once the portion of the itemcontaining the NA marker has been removed the item of interest, the NAmarker may isolated and/or prepared for PCR analysis utilizingtechniques known to those skilled in the art of PCR sample preparation.

Real-Time PCR Amplification

In many embodiments, the authentication process comprises amplifying thenucleic tag by polymerase chain reaction. However, conventional PCRamplification is not a quantitative detection method. Duringamplification, primer dimers and other extraneous nucleic acids areamplified together with the nucleic acid corresponding to the analyte.These impurities must be separated, usually with gel separationtechniques, from the amplified product resulting in possible losses ofmaterial. Although methods are known in which the PCR product ismeasured in the log phase, these methods require that each sample haveequal input amounts of nucleic acid and that each sample amplifies withidentical efficiency, and are therefore, not suitable for routine sampleanalyses. To allow an amount of PCR product to form which is sufficientfor later analysis and to avoid the difficulties noted above,quantitative competitive PCR amplification uses an internal controlcompetitor and is stopped only after the log phase of product formationhas been completed.

In a further development of PCR technology, real time quantitative PCRhas been applied to nucleic acid analytes or templates. In this method,PCR is used to amplify DNA in a sample in the presence of anonextendable dual labeled fluorogenic hybridization probe. Onefluorescent dye serves as a reporter and its emission spectra isquenched by the second fluorescent dye. The method uses the 5′ nucleaseactivity of Taq polymerase to cleave a hybridization probe during theextension phase of PCR. The nuclease degradation of the hybridizationprobe releases the quenching of the reporter dye resulting in anincrease in peak emission from the reporter. The reactions are monitoredin real time. Reverse transcriptase (RT)-real time PCR (RT-PCR) has alsobeen described (Gibson et al., 1996). Numerous commercially thermalcyclers are available that can monitor fluorescent spectra of multiplesamples continuously in the PCR reaction, therefore the accumulation ofPCR product can be monitored in ‘real time’ without the risk of ampliconcontamination of the laboratory. Heid, C. A.; Stevens, J.; Livak, K. L.;Williams, P. W. (1996). Real time quantitative PCR. Gen. Meth. 6:986-994.

In some embodiments of the anti-counterfeit authentication process, realtime PCR detection strategies may be used, including known techniquessuch as intercalating dyes (ethidium bromide) and other double strandedDNA binding dyes used for detection (e.g. SYBR green, a highly sensitivefluorescent stain, FMC Bioproducts), dual fluorescent probes (Wittwer,C. et al., (1997) BioTechniques 22: 176-181) and panhandle fluorescentprobes (i.e. molecular beacons; Tyagi S., and Kramer F R. (1996) NatureBiotechnology 14: 303-308). Although intercalating dyes and doublestranded DNA binding dyes permit quantitation of PCR productaccumulation in real time applications, they suffer from the previouslymentioned lack of specificity, detecting primer dimer and anynon-specific amplification product. Careful sample preparation andhandling, as well as careful primer design, using known techniques mustbe practiced to minimize the presence of matrix and contaminant DNA andto prevent primer dimer formation. Appropriate PCR instrument analysissoftware and melting temperature analysis permit a means to extractspecificity and may be used with these embodiments.

PCR amplification is performed in the presence of a non-primerdetectable probe which specifically binds the PCR amplification product,i.e., the amplified detector DNA moiety. PCR primers are designedaccording to known criteria and PCR may be conducted in commerciallyavailable instruments. The probe is preferably a DNA oligonucleotidespecifically designed to bind to the amplified detector molecule. Theprobe preferably has a 5′ reporter dye and a downstream 3′ quencher dyecovalently bonded to the probe which allow fluorescent resonance energytransfer. Suitable fluorescent reporter dyes include6-carboxy-fluorescein (FAM), tetrachloro-6-carboxy-fluorescein (TET),2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein (JOE) andhexachloro-6-carboxy-fluorescein (HEX). A suitable reporter dye is6-carboxy-tetramethyl-rhodamine (TAMRA). These dyes are commerciallyavailable from Perkin-Elmer, Philadelphia, Pa. Detection of the PCRamplification product may occur at each PCR amplification cycle. At anygiven cycle during the PCR amplification, the amount of PCR product isproportional to the initial number of template copies. The number oftemplate copies is detectable by fluorescence of the reporter dye. Whenthe probe is intact, the reporter dye is in proximity to the quencherdye which suppresses the reporter fluorescence. During PCR, the DNApolymerase cleaves the probe in the 5′-3′ direction separating thereporter dye from the quencher dye increasing the fluorescence of thereporter dye which is no longer in proximity to the quencher dye. Theincrease in fluorescence is measured and is directly proportional to theamplification during PCR. This detection system is now commerciallyavailable as the TaqMan® PCR system from Perkin-Elmer, which allows realtime PCR detection.

In an alternative embodiment, the reporter dye and quencher dye may belocated on two separate probes which hybridize to the amplified PCRdetector molecule in adjacent locations sufficiently close to allow thequencher dye to quench the fluorescence signal of the reporter dye. Aswith the detection system described above, the 5′-3′ nuclease activityof the polymerase cleaves the one dye from the probe containing it,separating the reporter dye from the quencher dye located on theadjacent probe preventing quenching of the reporter dye. As in theembodiment described above, detection of the PCR product is bymeasurement of the increase in fluorescence of the reporter dye.

Molecular beacons systems are frequently used with real time PCR forspecifically detecting the nucleic acid template in the samplequantitatively. For instance, the Roche Light Cycler™ or other suchinstruments may be used for this purpose. The detection moleculeconfigured to the molecular beacon probe may be visible under daylightor conventional lighting and/or may be fluorescent. It should also benoted that the detection molecule may be an emitter of radiation, suchas a characteristic isotope.

The ability to rapidly and accurately detect and quantify biologicallyrelevant molecules with high sensitivity is a central issue for medicaltechnology, national security, public safety, and civilian and militarymedical diagnostics. Many of the currently used approaches, includingenzyme linked immunosorbant assays (ELISAs) and PCR are highlysensitive. However, the need for PCR amplification makes a detectionmethod more complex, costly and time-consuming. In certain embodimentsanti-counterfeit nucleic acid tags are detected by Surface EnhancedRaman Scattering (SERS) as described in U.S. Pat. No. 6,127,120 byGraham et al. SERS is a detection method which is sensitive torelatively low target (nucleic acid) concentrations, which canpreferably be carried out directly on an unamplified samples. Nucleicacid tags and/or nucleic acid probes can be labeled or modified toachieve changes in SERS of the nucleic acid tag when the probe ishybridized to the nucleic acid tag. The use of SERS for quantitativelydetecting a nucleic acid provides a relatively fast method of analyzingand authenticating a particular product.

Another detection method useful in the invention is theQuencher-Tether-Ligand (QTL) system for a fluorescent biosensordescribed in U.S. Pat. No. 6,743,640 by Whitten et al. The QTL systemprovides a simple, rapid and highly-sensitive detection of biologicalmolecules with structural specificity. QTL system provides a chemicalmoiety formed of a quencher (Q), a tethering element (T), and a ligand(L). The system is able to detect target biological agents in a sampleby observing fluorescent changes.

The QTL system can rapidly and accurately detect and quantify targetbiological molecules in a sample. Suitable examples of ligands that canbe used in the polymer-QTL approach include chemical ligands, hormones,antibodies, antibody fragments, oligonucleotides, antigens,polypeptides, glycolipids, proteins, protein fragments, enzymes, peptidenucleic acids and polysaccharides. Examples of quenchers for use in theQTL molecule include methyl viologen, quinones, metal complexes,fluorescent dyes, and electron accepting, electron donating and energyaccepting moieties. The tethering element can be, for example, a singlebond, a single divalent atom, a divalent chemical moiety, and amultivalent chemical moiety. However, these examples of the ligands,tethering elements, and quenchers that form the QTL molecule are not tobe construed as limiting, as other suitable examples would be easilydetermined by one of skill in the art.

Kits for Authenticating Items Using Nucleic Acid-Linked OpticalReporters

The invention also provides kits for authenticating items of interestusing the methods of the invention. The kits of the invention maycomprise, for example, a container of the optical reporter marker, and asample tube for holding a collected sample of the item or item to beauthenticated. The kits may further comprise an applicator for applyinga sample of the optical reporter to the item. The kits may still furthercomprise a collection tool for taking a sample of the labeled item fortransfer to the sample tube. The kits may further comprise a portablelight source for detecting the optical reporters.

By way of example, the optical reporter marker may be in the form of aliquid solution or dispersion, and the container with the kit would besuitably configured for holding a liquid. The applicator of the kit maycomprise an “eye-dropper” for applying liquid optical reporter markersolution to the item in droplet form, a spatula for smearing thesolution on an item, a syringe for injecting the solution into an item,or like type of applicator. The collection tool of the kit may comprisea spoon, gouge, a scraping or abrading tool for removing a sample of thelabeled item, a blade or scissors for cutting a piece of the item, acloth (which may be solvent-moistened) for wiping a sample from theitem, or the like. The sample tube of the kit may comprise a sealablevial or eppendorf tube, and may contain solvent or solution forextraction of the optical reporter marker from the sample taken from thetagged item. The portable light source of the kit may comprise ahand-held UV lamp suitable for detecting the optical reporter marker.

The kit may further comprise primers and/or probes as well as solutionsappropriate for PCR analysis. The kit may further comprise a small PCRinstrument for analysis of the extracted optical reporter marker.

The kits of the invention thus provide a convenient, portable system forpracticing the methods of the invention.

Synthesis of UCP Particles Covalently Linked to Biomolecules

Nucleotide-labeled optical reporters in accordance with the inventioncan be made by a variety of methods, including those depicted in theco-pending U.S. application “Methods for linking Optical Reporters toBiomolecules,” which is herein incorporated by reference.

Preferred methods for preparing UCP particles covalently linked to DNAare provided in the following Examples.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

Efforts have been made to ensure accuracy with respect to numbers used(e.g. amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees Centigrade, and pressure is at or nearatmospheric.

Example 1 Printer Ink Cartridges

With a demand for higher durability and higher image quality in recentyears, printers have made significant advances. The quality of printersand ink are superior having good weatherability, optical density and thelike. Most printers for home or small offices require the use ofconsumables such as ink cartridges and toner, which must be replacedfrom time to time as the ink wears out.

FIG. 3 shows an exemplary printer which uses an ink cartridge or toner.The schematic constitution of a mechanism portion of an ink jet printer300 will be described below. A printer main body is constituted of asheet feeding portion, a sheet conveying portion, a carriage portion, asheet discharge portion, and a cleaning portion, and an externalpackaging portion for protecting them and providing them with a design,each of which plays a role of each mechanism. The printer 300 comprisesa carriage portion 302, which is show in FIG. 4, which is detachablymounted to its ink toner cartridge 304.

In one embodiment, the liquid or powder ink itself will contain thenucleic acid material. In other words, liquid ink containing variouswater-soluble organic solvents usually used in ink jet inks may becombined with the nucleic acid material for verification purposes. Inthe present invention, the ink may further be incorporated with variousadditives such as a surfactant, a pH adjuster, a rust inhibitor, anantiseptic, a mildew proofing agent, an antioxidant, areduction-preventive agent, an evaporation accelerator, a chelatingagent and a water-soluble polymer. Thus, when the printer is used, theresulting printed paper will contain ink from the printer whichcomprises the nucleic acid material. For verification purposes, a pieceof paper can be tested to determine whether or not the ink used to printit was used with genuine ink. This technique may also be used todetermine the source of the ink, for investigative purposes. Further, ifliquid ink is not used, but other fine particles are used for eitherplain printing or producing glossy, transparency, colored materials, andthe like, the fine particles may also be combined with the nucleic acidmaterial in a similar fashion.

In another embodiment, it is the cartridge toner, i.e., the carbon tonerwhich comprises the nucleic acid material. The carriage portion 302 inFIG. 4 has a corresponding ink toner cartridge 304 for carrying outprinting functions with an ink storage portion for storing the ink. Ifthe cartridge toner 304 contains the nucleic acid material (not theink), then any resulting item produced by the printer will necessarilyhave the genetic material. So when sheets of paper are fed into theprinter, as soon as an image is formed in the paper as it goes throughthe rolling guides, the cartridge combines, at this point, the ink fromthe cartridge toner and the nucleic acid material, delivering the finalitem, the printed paper with the DNA.

Example 2

Up-converting phosphor nanopowder (doped yttrium oxide and yttriumoxysulfide upconverting particles) were obtained from Nanocerox, Inc.,Ann Arbor, Mich.

ABBREVIATIONS

UCP Up converting phosphor

UTP Up converting phosphor technology

OpR optical reporter particle

cOpR coated optical reporter particle

TEOS tetraethoxysilane, tetraethyl orthosilicate; ethyl

silicate; silicic acid, tetraethyl ester; or silicon ethoxide

MOS methyl oxysilane

EOS ethyl oxysilane

POS propyl oxysiline

NHS N-Hydroxsuccinimde

IOA Iodoacetamide

DIPCI Diisopropylcarbodiimide

DCM dichloromethane/methylene chloride

DIPEA diisopropyl ethylamine

DMF N,N-dimethylformamide

DMAP 4-dimethylaminopyridine

ECDI 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide

EtOAc ethyl acetate

EtOH ethanol

hplc high performance liquid chromatography

mCPBA m-chloroperbenzoic acid

MeCN acetonitrile

TLC thin layer chromatography

Doped Yttrium Oxysulfide with Oxypropylsulfanylacetamide-Linked DNA

The synthetic procedure of this Example is shown below in Scheme A.

This example demonstrates that the compositions produced by the methodsof the inventions, particularly those methods in which nucleotides arelinked to a phosphor, that the nucleotide attached to the compositioncan be detected directly by techniques such as PCR. The phosphorcompound utilized in this example was Yttrium oxysulfide up convertingparticles as well as an amine linked DNA oligomer.

Detection of Bound DNA to Phosphor Particles by Real-Time-PCR.

The equipment and supplies utilized for RT-PCR were the following. PCRcapillary system (20 ul capillary) by Roche Diagnostics, LightCycler 2by Roche Diagnostics, SYBR Green ReadyMix RT-PCR kit by Sigma-Aldrichand SYBR Green JumpStart Tag mix by Sigma.

The following primers were specifically designed for amplification ofthe DNA oligomer attached to the phosphor particles produced by themethods of the invention. Primer 1-(5′-CGCCAGGGT TTTCCCAGTCACGAC-3′) andPrimer 2 (5′-CAGGAAACAGCTATGAC-3′). The final concentration of theprimers for RT-PCR amplification was 0.05 uM in the RT-PCR r×n sample.The size of the amplicon generated during RT-PCR with this primer pairwas approximately 150 bp in length.

The RT-PCR run conditions were as follows. One pre-heating cycle of 95°C. for 5 minutes, followed by 40 cycles of 20 seconds at 95° C., 40seconds at the annealing temperature of 50° C., with polymeraseextension at 72° C. for 20 seconds.

The isolated phosphor particles containing DNA molecules wereresuspended in and diluted 1/10, 1/100, and 1/1000, respectively forRT-PCR analysis. Each RT-PCR sample contained 15 ul of RT-PCR master mix0.5 ul of each Primer stock solution, 1 ul of a specified dilutedphosphor containing DNA sample, and 13 ul water were mixed and put into20 ul capillary tubes. Positive and Negative controls were alsoprepared. Duplicates of all RT-PCR samples were prepared and analyzed.

The results from the RT-PCR experiment where similar to those shown inFIG. 5, discussed below in Example 3.

Example 3 Doped Yttrium Oxysulfide with (oxy-propylamino)-acetic acid5-amino-4-oxo-pentyl ester-linked DNA

The synthetic procedure of this example is shown below in Scheme B.

This example demonstrates that the compositions produced by the methodsof the inventions, particularly those methods in which nucleotides arelinked to a phosphor, that the nucleotide attached to the compositioncan be detected directly by techniques such as PCR. The phosphorcompound utilized in this example was Yttrium oxysulfide up convertingparticles as well as an amine linked DNA oligomer.

Detection of Bound DNA to Phosphor Particles by Real-Time-PCR.

The equipment and supplies utilized for RT-PCR were the following. PCRcapillary system (20 ul capillary) by Roche Diagnostics, LightCycler 2by Roche Diagnostics, SYBR Green ReadyMix RT-PCR kit by Sigma-Aldrichand SYBR Green JumpStart Tag mix by Sigma.

The following primers were specifically designed for amplification ofthe DNA oligomer attached to the phosphor particles produced by themethods of the invention. Primer 1-(5′-CGCCAGGGT TTTCCCAGTCACGAC-3′) andPrimer 2 (5′-CAGGAAACAGCTATGAC-3′). The final concentration of theprimers for RT-PCR amplification was 0.05 uM in the RT-PCR r×n sample.The size of the amplicon generated during RT-PCR with this primer pairwas approximately 150 bp in length.

The RT-PCR run conditions were as follows. One pre-heating cycle of 95°C. for 5 minutes, followed by 40 cycles of 20 seconds at 95° C., 40seconds at the annealing temperature of 50° C., with polymeraseextension at 72° C. for 20 seconds.

The isolated phosphor particles containing DNA molecules wereresuspended in and diluted 1/10, 1/100, and 1/1000, respectively forRT-PCR analysis. Each RT-PCR sample contained 15 ul of RT-PCR master mix0.5 ul of each Primer stock solution, 1 ul of a specified dilutedphosphor containing DNA sample, and 13 ul water were mixed and put into20 ul capillary tubes. Positive and Negative controls were alsoprepared. Duplicates of all RT-PCR samples were prepared and analyzed.

The results from the RT-PCR experiment are shown in FIG. 5. The resultsin FIG. 5 show that the 1/100 dilution sample had a Ct of 30, while the1/10 and 1/1000 dilution had a Ct of 33 and Ct of 36, respectively. Atthe 1/10 dilution the concentration of the UCP particles is high enoughto quench the PCR signal, thus delaying the cycle in which amplificationof the target DNA is present.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. An authentication method for authenticating an ink within an inkcartridge, said method comprising the steps of: applying a particularnucleic acid material associated with a particular sequence of nucleicacid bases to the ink; collecting a sample of the ink having the nucleicacid; and verifying whether the ink is genuine by detecting saidparticular nucleic acid material.
 2. The method of claim 1 wherein theparticular nucleic acid material is deoxy-ribo nucleic acid (DNA). 3.The method of claim 1 wherein the particular nucleic acid material isribonucleic acid (RNA).
 4. The method of claim 1 further comprisingdetecting the particular nucleic acid by performing a polymerase chainreaction (PCR) of the nucleic acid material.
 5. An authentication methodfor authenticating a toner compound within a toner housing, said methodcomprising the steps of: applying a particular nucleic acid materialassociated with a particular sequence of nucleic acid bases to the tonercompound; collecting a sample of the toner compound having the nucleicacid; and verifying whether the toner compound is genuine by detectingsaid particular nucleic acid material.
 6. The method of claim 1 whereinthe particular nucleic acid material is deoxy-ribo nucleic acid (DNA).7. The method of claim 1 wherein the particular nucleic acid material isribonucleic acid (RNA).
 8. The method of claim 1 further comprisingdetecting the particular nucleic acid by performing a polymerase chainreaction (PCR) of the nucleic acid material.
 9. The method forauthenticating ink in an ink cartridge comprising the steps of;providing an optical reporter marker, the optical reporter marker havingat least one light emitting upconverting phosphor particle linked to atleast one nucleic acid material, the nucleic acid material having anidentifiable portion, introducing the optical reporter marker to the inkof interest, detecting the optical reporter marker associated with theink with a light source, obtaining a sample of the optical reportermarker from the ink of interest for analysis; and analyzing thecollected sample to detect the presence of the identifiable portion ofthe nucleic acid material linked to the upconverting phosphor particle.10. The method of claim 9, wherein the optical reporter marker has thecomposition of the formula I:(cOpR)-[L-(NA)]_(m)  I wherein: m is an integer greater than 1; (cOpR)is a coated optical reporter particle; (NA) is a nucleic acid oligomerof the nucleic acid material of detectable sequence; and L is a linkinggroup covalently bound to the coated optical reporter particle and tothe nucleic acid oligomer.
 11. The method of claim 10, wherein (cOpR)comprises an upconverting phosphor (UCP) material.
 12. The method ofclaim 10, wherein (NA) is a single or double stranded DNA moleculehaving a length of between about 40 base pairs and about 1000 basepairs.
 13. The method of claim 10, wherein L comprises an alkylenemoiety having a first end covalently bound to the coated opticalreporter particle and a second end covalently bound to the nucleic acidoligomer.
 14. The method of claim 11, wherein (UCP) is an upconvertingphosphor particle of the formula:Y_(x)Yb_(y)Er_(z)O₂S; orNa(Y_(x)Yb_(y)Er_(z))F₄; wherein: x is from about 0.6 to about 0.95; yis from about 0.05 to about 0.35; and z is from about 0.1 to about0.001.
 15. The method of claim 10, wherein L is of the formula:-A-R¹—B— wherein: R¹ is C₂₋₈alkylene; -A- is a group covalently bondedto the surface of the coated optical reporter; and —B— is a groupcovalently bonded to the 3′ or 5′ end of the nucleic acid oligomer. 16.A method for authenticating ink comprises the steps of; providing anoptical reporter marker, the optical reporter marker having at least onelight emitting upconverting phosphor particle linked to at least onenucleic acid taggant, the nucleic acid taggant having an identifiableportion, introducing the optical reporter marker to the ink, detectingthe optical reporter marker associated with the ink with a light source,obtaining a sample of the optical reporter marker from the ink foranalysis; and analyzing the collected sample to detect the presence ofthe identifiable portion of the nucleic acid taggant linked to theupconverting phosphor particle.
 17. The method of claim 16, wherein theoptical reporter marker has the composition of the formula I:(cOpR)-[L-(NA)]_(m)  I wherein: m is an integer greater than 1; (cOpR)is a coated optical reporter particle; (NA) is a nucleic acid oligomerof detectable sequence; and L is a linking group covalently bound to thecoated optical reporter particle and to the nucleic acid oligomer. 18.The method of claim 17, wherein (cOpR) comprises an upconvertingphosphor (UCP) material.
 19. The method of claim 17, wherein (NA) is asingle or double stranded DNA molecule having a length of between about40 base pairs and about 1000 base pairs.
 20. The method of claim 17,wherein L comprises an alkylene moiety having a first end covalentlybound to the coated optical reporter particle and a second endcovalently bound to the nucleic acid oligomer.
 21. The method of claim18, wherein (UCP) is an upconverting phosphor particle of the formula:Y_(x)Yb_(y)Er_(z)O₂S; orNa(Y_(x)Yb_(y)Er_(z))F₄; wherein: x is from about 0.6 to about 0.95; yis from about 0.05 to about 0.35; and z is from about 0.1 to about0.001.
 22. The method of claim 17, wherein L is of the formula:-A-R¹—B— wherein: R¹ is C₂₋₈alkylene; -A- is a group covalently bondedto the surface of the coated optical reporter; and —B— is a groupcovalently bonded to the 3′ or 5′ end of the nucleic acid oligomer. 23.The method of claim 22, wherein -A- is —O—.
 24. The method of claim 22,wherein —R¹— is —(CH₂)_(n)— and wherein n is from 2 to
 8. 25. The methodof claim 22, wherein —B— is: —S—; —O—; —NR^(a)—; —S—(CH₂)_(p)—;—O—(CH₂)_(p)—; —NR^(a)—(CH₂)_(p)—; —S—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—;—O—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—;—NR^(a)—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—;—S—C(O)—(CH₂)_(r)C(O)—NR^(a)—(CH₂)_(p)—;—O—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p); or—NR^(a)—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—; wherein: p is from 2 to8; q is from 1 to 8; r is from 2 to 8; and each R^(a) is independentlyhydrogen or C₁₋₆alkyl;
 26. The method of claim 22, wherein —B— is:—S—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p) or—NR^(a)—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—; wherein p, q, r and R^(a)are as recited in claim
 9. 27. The method of claim 26, wherein: p isfrom 2 to 6; q is from 1 to 3; and r is 2 or
 3. 28. The method of claim22, wherein —B— is: —S—CH₂—C(O)—NH—(CH₂)₆—; or—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₆—;
 29. The method of claim 17, wherein thecOpR is coated with silica.
 30. The method of claim 29, wherein thesilica comprises at least one Si—O bond.
 31. The method of claim 16,wherein the optical reporter marker has the composition of the formulaII:(UCP)-[A-R¹—X—R²—C(O)—NR^(a)—R³-(DNA)]_(m)  II wherein: m is an integergreater than 1; UCP is an upconverting phosphor particle; DNA is asingle or double stranded deoxyribonucleic acid oligomer; -A- is a groupcapable of covalently bonding to the surface of the Upconvertingphosphor particle; R¹ is C₂₋₈alkylene, R² is C₁₋₈alkylene or—C(O)—C₁₋₈alkylene-; —X— is —O—, —S— or —NR^(a)—; R³ is C₂₋₈alkylene;and R^(a) is hydrogen or C₁₋₆alkyl.
 32. The composition of claim 31,wherein the composition is of the formula IV:(UCP)-[O—(CH₂)_(s)—S—(CH₂)_(t)—C(O)—NH—(CH₂)_(v)-(DNA)]_(m)  IV wherein:is from 2 to 6; v is from 2 to 6; t is from 1 to 3; and m, UCP and DNAare as recited in claim
 30. 33. The composition of claim 31, wherein thecomposition is of the formula V:(UCP)-[O—(CH₂)_(s)—NH—C(O)—(CH₂)_(u)—C(O)—NH—(CH₂)_(v)-(DNA)]_(m)  Vwherein: s is from 2 to 6; v is from 2 to 6; u is 2 or 3; and m, UCP andDNA are as recited in claim
 30. 34. The composition of claim 31, whereinthe composition is of the formula VI:(UCP)-[O—(CH₂)₃—S—CH₂—C(O)—NH—(CH₂)₆-(DNA)]_(m)  VI wherein m, UCP andDNA are as recited in claim
 15. 35. The composition of claim 31, whereinthe composition is of the formula VII:(UCP)-[O—(CH₂)₃—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₆-(DNA)]_(m)  VII wherein m,UCP and DNA are as recited in claim
 15. 36. The composition of claim 17,wherein the cOpR comprises a visually detectable light emitting materialselected from the group consisting of a fluorescent dye, a upconvertingphosphor, a ceramic powder, or a quantum dot.
 37. The composition ofclaim 36, where said light emitting materials are excitable by UV or aninfrared light source.
 38. The method of claim 17, wherein the cOpRcomprises at least one electromagnetic emitting material.
 39. Thecomposition of claim 38, where the electromagnetic emitting material isdetectable by devices which provide sources selected from the groupconsisting of an infrared radiation source, magnetic field source, aquantum dot or electromagnetic pulse.
 40. The method of claim 16,wherein detecting the nucleic acid taggant comprises polymerase chainreaction analysis.
 41. The method of claim 16, wherein the nucleic acidtaggant is ds DNA.
 42. The method of claim 16, wherein said associatingthe optical reporter marker with the ink further comprises a databasemanagement system that associates the specific optical reporter markerwith said article of interest.
 43. A method for authenticating a ink,comprising: providing a optical reporter marker having at least onenucleic acid taggant; applying the marker compound to at least one ink,which enters at least one supply chain; locating and collecting a sampleof the optical reporter marker from said ink after said ink has enteredsaid supply chain; and identifying said nucleic acid product in saidink.
 44. A kit for authenticating ink comprising: a container of opticalreporter marker; and an applicator for applying a sample of the opticalreporter to the ink.